The morbid genome of ciliopathies: an update.

PURPOSE: Ciliopathies are highly heterogeneous clinical disorders of the primary cilium. We aim to characterize a large cohort of ciliopathies phenotypically and molecularly. METHODS: Detailed phenotypic and genomic analysis of patients with ciliopathies, and functional characterization of novel candidate genes. RESULTS: In this study, we describe 125 families with ciliopathies and show that deleterious variants in previously reported genes, including cryptic splicing variants, account for 87% of cases. Additionally, we further support a number of previously reported candidate genes (BBIP1, MAPKBP1, PDE6D, and WDPCP), and propose nine novel candidate genes (CCDC67, CCDC96, CCDC172, CEP295, FAM166B, LRRC34, TMEM17, TTC6, and TTC23), three of which (LRRC34, TTC6, and TTC23) are supported by functional assays that we performed on available patient-derived fibroblasts. From a phenotypic perspective, we expand the phenomenon of allelism that characterizes ciliopathies by describing novel associations including WDR19-related Stargardt disease and SCLT1- and CEP164-related Bardet-Biedl syndrome. CONCLUSION: In this cohort of phenotypically and molecularly characterized ciliopathies, we draw important lessons that inform the clinical management and the diagnostics of this class of disorders as well as their basic biology.

A cohort of 17 patients with kyphoscoliotic Ehlers-Danlos syndrome caused by biallelic mutations in FKBP14: expansion of the clinical and mutational spectrum and description of the natural history.

PurposeIn 2012 we reported in six individuals a clinical condition almost indistinguishable from PLOD1-kyphoscoliotic Ehlers-Danlos syndrome (PLOD1-kEDS), caused by biallelic mutations in FKBP14, and characterized by progressive kyphoscoliosis, myopathy, and hearing loss in addition to connective tissue abnormalities such as joint hypermobility and hyperelastic skin. FKBP14 is an ER-resident protein belonging to the family of FK506-binding peptidyl-prolyl cis-trans isomerases (PPIases); it catalyzes the folding of type III collagen and interacts with type III, type VI, and type X collagens. Only nine affected individuals have been reported to date.MethodsWe report on a cohort of 17 individuals with FKBP14-kEDS and the follow-up of three previously reported patients, and provide an extensive overview of the disorder and its natural history based on clinical, biochemical, and molecular genetics data.ResultsBased on the frequency of the clinical features of 23 patients from the present and previous cohorts, we define major and minor features of FKBP14-kEDS. We show that myopathy is confirmed by histology and muscle imaging only in some patients, and that hearing impairment is predominantly sensorineural and may not be present in all individuals.ConclusionOur data further support the extensive clinical overlap with PLOD1-kEDS and show that vascular complications are rare manifestations of FKBP14-kEDS.

Kyphoscoliotic type of Ehlers-Danlos Syndrome (EDS VIA) in six Egyptian patients presenting with a homogeneous clinical phenotype.

UNLABELLED: The kyphoscoliotic type of the Ehlers-Danlos syndrome (EDS VIA) is a rare recessively inherited connective tissue disorder characterized by bruisable, hyperextensible skin, generalized joint laxity, severe muscular hypotonia at birth and progressive congenital scoliosis or kyphosis. Deficiency of the enzyme lysyl hydroxylase 1 (LH1) due to mutations in PLOD1 results in underhydroxylation of collagen lysyl residues and, hence, in the abnormal formation of collagen cross-links. Here, we report on the clinical, biochemical, and molecular findings in six Egyptian patients from four unrelated families severely affected with EDS VIA. In addition to the frequently reported p.Glu326_Lys585dup, we identified two novel sequence variants p.Gln208* and p.Tyr675*, which lead either to loss of function of LH1 or to its deficiency. All affected children presented with similar clinical features of the disorder, and in addition, several dysmorphic craniofacial features, not yet described in EDS VIA. These were specific for the affected individuals of each family, but absent in their parents and their unaffected siblings. CONCLUSION: Our description of six patients presenting with a homogeneous clinical phenotype and dysmorphic craniofacial features will help pediatricians in the diagnosis of this rare disorder.

Acromesomelic Dysplasia With Homozygosity for a Likely Pathogenic BMPR1B Variant: Postaxial Polydactyly as a Novel Clinical Finding.

BACKGROUND: Acromesomelic chondrodysplasias are a rare subgroup of the clinically and genetically heterogeneous osteochondrodysplasias that are characterised by abnormalities in the limb development and short stature. Here, we report a 2-year-old boy, offspring of consanguineous parents, with acromesomelic dysplasia and postaxial polydactyly in which exome sequencing identified a novel homozygous missense variant in BMPR1B. The patient showed skeletal malformation of both hands and feet that included complex brachydactyly with the thumbs most severely affected, postaxial polydactyly of both hands, shortened toes as well as a bilateral hypoplasia of the fibula. METHODS: Whole trio exome sequencing was conducted to identify potential genetic variants in the patient. RESULTS: The analysis identified the biallelic variant NM_001203.3:c.821A > G;p.(Gln274Arg) in BMPR1B, a gene encoding bone morphogenetic protein receptor 1B. CONCLUSION: The skeletal phenotype can be brought in line with the phenotypes of previously reported cases of BMPR1B-associated chondrodysplasias. However, the postaxial polydactyly described here is a novel clinical finding in a BMPR1B-related case; notably, it has previously been reported in other acromesomelic dysplasia cases caused by homozygous pathogenic variants in GDF5-a gene which encodes for growth differentiation factor 5, a high-affinity ligand to BMPR1B.

Detection of Heterozygous Carriers of PKU in Egypt: Successful Application of a Simple Biochemical Method.

The absence of a convenient, direct enzymatic assay for detecting phenylketonuria (PKU) heterozygotes together with the difficulty of the molecular testing due to the large number of mutations in the phenylalanine hydroxylase (PAH) gene has resulted in continued effort to develop an accurate procedure to discriminate the heterozygous individuals from the homozygous normal population. Aiming to find out a method that is simple and reliable for PKU carrier screening, we compared the biochemical data of 20 known PKU obligate heterozygotes with those of 45 presumed normal homozygous controls. Fasting blood samples from all subjects were analyzed for plasma phenylalanine and tyrosine using an amino-acid analyzer. Micromolar plasma concentrations of phenylalanine and tyrosine in addition to Phe/Tyr and Phe2/Tyr ratios were determined and statistical analysis of the difference between the two groups was done using the student's t test. Mean values for phenylalanine concentrations, Phe/Tyr and Phe2/Tyr ratios were significantly higher in PKU heterozygotes than in control subjects. In addition, ROC curve analysis was performed for the same four biochemical variables. The value for the area under the curve (ROCAUC) was obtained for each parameter with the Phe2/Tyr ratio having an area of 1, which means that it had perfect discrimination. When the ratio Phe/Tyr was plotted against the Phe2/Tyr, all the studied control subjects and none of the PKU carriers fell below the values 1.2 and 80, respectively. Finally, by applying the same graphic plot for 20 at-risk PKU family members asking for premarital carrier testing, nine PKU heterozygotes were detected. The same results were successfully reproduced using the values obtained from the ROC curve analysis, indicating a high degree of accuracy for this screening method. In conclusion, the used biochemical method is simple and reliable and it can be useful in the widespread screening for PKU carriers.

Overlap between Fibular Aplasia, Tibial Campomelia, and Oligosyndactyly and Fuhrmann's Syndromes in an Egyptian Female Infant.

We report a rare congenital limb defect with combined features of both fibular aplasia, tibial campomelia, and oligosyndactyly (FATCO) and Fuhrmann's syndromes. A female newborn infant, born to nonconsanguineous Egyptian parents, presented with isolated abnormalities of the lower limbs comprising bilateral shortening and anterior bowing of the lower limbs at the distal third of the tibia and split foot. Radiographic examination revealed complete absence of both fibulae, anterolateral bowing and shortening of the tibia, bowing of the femora, and absence of several metatarsal and phalangeal bones. The upper limbs were clinically and radiologically normal, and the infant had neither facial dysmorphism nor other associated visceral anomalies. The presented case highlights an extremely rare limb deficiency syndrome, and together with additional case reports, it could be useful to further delineate this condition.

Novel PITX2 gene mutations in patients with Axenfeld-Rieger syndrome.

PURPOSE: Mutations in the bicoid-like transcription factor PITX2 gene often result in Axenfeld-Rieger syndrome (ARS), an autosomal-dominant inherited disorder. We report here the discovery and characterization of novel PITX2 deletions in a small kindred with ARS. METHODS: Two familial patients (father and son) from a consanguineous family were examined in the present study. Patient DNA samples were screened for PITX2 mutations by DNA sequencing and for copy number variation by SYBR Green quantitative polymerase chain reaction (PCR) analysis. RESULTS: We report a novel deletion involving the coding region of PITX2 in both patients. The minimum size of the deletion is 1 421 914 bp that spans one upstream regulatory element (CE4), PITX2 and a minimum of 13 neighbouring genes. The maximum size of the deletion is 3 789 983 bp. The proband (son) additionally possesses a novel 2-bp deletion in a non-coding exon of the remaining PITX2 allele predicted to alter correct splicing. CONCLUSION: Our findings implicate a novel deletion of the PITX2 gene in the pathogenesis of ARS in the affected family. This ARS family presented with an atypical and extremely severe phenotype that resulted in four miscarriages and the death at 10 months of age of a sib of the proband. As the phenotypic manifestations in the proband are more severe than that of the father, we hypothesize that the deletion of the entire PITX2 allele plus a novel 2-bp deletion (observed in the proband) within the remaining PITX2 allele together contributed to the atypical ARS presentation in this family. This is the first study reporting on bi-allelic changes of PITX2 potentially contributing to a more severe ARS phenotype.

De novo EDA mutations: Variable expression in two Egyptian families.

OBJECTIVE: Mutations in the EDA gene, encoding the epithelial morphogen ectodysplasin-A, can result in different but overlapping phenotypes. Therefore the aim of the study was to search for etiological variations of EDA and other candidate genes in two unrelated Egyptian male children with sporadic non-syndromic tooth agenesis (NTA) and hypohidrotic ectodermal dysplasia (HED). DESIGN: Direct sequencing of the coding regions including exon-intron boundaries of EDA, MSX1, PAX9, WNT10A and EDAR was performed in probands and their available family members. RESULTS: Two etiological mutations were found in the EDA coding region. The patient with NTA in both deciduous and permanent dentition was a carrier of a novel in-frame deletion situated in the short collagenous domain (c.663-680delTCCTCCTGGTCCTCAAGG, p.222-227delPPGPQG). The second mutation, located outside the minimal furin consensus motif (c.463C>T, p.Arg155Cys, rs132630312), was identified in the patient exhibiting all typical features of HED. The identified EDA mutations were not detected in probands' family members as well as in 188 unrelated control individuals. No pathogenic variants were found in the MSX1, PAX9, WNT10A and EDAR genes. CONCLUSION: Our results increase the knowledge of the spectrum of EDA mutations and confirm that this gene is an important candidate gene for two developmental diseases sharing the common feature of the congenital lack of teeth. In addition, these results can support the hypothesis that X-linked HED and EDA-related NTA are the same disease with different degrees of severity.

Postmortem clinical examination by experienced clinical geneticists as an alternative to conventional autopsy for assessment of fetal and perinatal deaths in countries with limited resources.

The aim of this study was to investigate the usefulness of postmortem external examination performed by an experienced clinical geneticist as an alternative to autopsy in countries with limited resources. We studied a consecutive cohort of couples seeking genetic counseling for fetal loss or perinatal death over a period of 3 years. The study involved 230 couples; only 57 of them submitted a fetus or dead neonate, for whom a meticulous postmortem clinical examination was performed by an experienced clinical geneticist. The diagnosis rate for the group of cases subjected to postmortem examination (57.9%) was much higher than that of the group that comprised cases for which diagnosis was made through evaluation of medical records (27.2%). Whenever fetal or neonatal autopsy is refused or is not feasible, a comprehensive fetal or perinatal postmortem external examination by an experienced clinical geneticist may be a reasonable substitute.

ß-Globin Mutations in Egyptian Patients With ß-Thalassemia.

ß-thalassemia is a common hereditary disorder, particularly in Middle Eastern countries. More than 200 mutations in the ß globin gene have been reported; most are point mutations in functionally important regions (HBB; OMIM #141900)). The spectrum of mutations varies significantly between different geographical regions; only a few common mutations of ß-globin cause ß-thalassemia in each population. The aim of this study was to determine the spectrum of mutations that cause ß-thalassemia in the North Coast of Egypt and to investigate their correlation with the phenotypic severity of ß-thalassemia. We carried out our study with a total of 47 Egyptian patients (25 male and 22 female) confirmed to have ß-thalassemia. Evaluation of ß-thalassemia mutations revealed the presence of 10 ß-globin mutations. The most frequently encountered mutations were intronic: IVS 1.6 [T>C] (27.66%) and IVS 1.110 [G>A] (22.35%), followed by IVS 2.848 [C>A], IVS 1.1 (G>A), and IVS 2.745 [C>G]. We observed the exonic and promoter mutations less frequently. A homozygous mutation was found in 24 patients (51%) and compound heterozygous mutations were found in 13 patients (28%). However, in 9 patients (19%), we identified only 1 mutation. In 1 patient (2%), we detected no mutation. The detection rate of the method that we used in our population was 88% (83 of the tested 94 alleles). The results we obtained did not reveal any correlation between genotype and phenotype among patients with ß-thalassemia.

A plakophilin-1 gene mutation in an egyptian family with ectodermal dysplasia-skin fragility syndrome.

Ectodermal dysplasia-skin fragility syndrome (ED-SFS) is a rare genodermatosis caused by mutations in the PKP1 gene, encoding the desmosomal plaque protein plakophilin-1. Since its initial description in 1997, few individuals with this disorder have been reported to date. Here, we present the first Egyptian cases of ED-SFS, carrying a novel homozygous mutation in the PKP1 gene. Direct sequencing of the amplified DNA from the affected cases disclosed a G-to-T transversion at nucleotide position c.203-1 within intron 1 of PKP1 (c.203-1G>T). To the best of our knowledge, this mutation has not been previously described in the databases.

Screening for the mitochondrial A1555G mutation among Egyptian patients with non-syndromic, sensorineural hearing loss.

BACKGROUND & AIM: Hearing loss is the most frequent form of neurosensory deficit in humans. Although the majority of hereditary hearing loss is due to nuclear gene mutations, it has become clear the significant contribution of mitochondrial genes. The first mitochondrial mutation shown to cause non-syndromic hearing loss in humans was the A1555G mutation in the small ribosomal RNA gene (12S rRNA). It has been detected in hundreds of families of different ethnic backgrounds, making it one of the prevalent genetic causes of hearing loss currently identified. However, there are major differences between ethnic groups regarding the frequency of this mutation. Few studies have been made in Arab countries, especially in Egypt. Here we report the prevalence of the mitochondrial mutation A1555G among patients with non-syndromic hearing loss (NSHL) and in healthy individuals with normal hearing in the Egyptian population. SUBJECTS & METHODS: The study was conducted on 97 patients with SNHL and 300 unrelated healthy Egyptian individuals, with normal hearing, as normal control subjects. Polymerase chain reaction followed by restriction enzyme digestion was used to screen the DNA samples of all subjects for the A1555G mutation. RESULTS: Participants included 97 cases with SNHL, 46 males and 51 females. Their ages ranged from 1 month to 65 years with the mean age 6.2 years (SD ± 8.2). Paternal consanguinity was reported in 46% (35/76) of the studied families. The A1555G mutation was found in one of the 97 patients (1.3%), while it has not been detected in the 300 control samples. CONCLUSION: Our findings indicate that, even in absence of exposure to aminoglycosides, the mitochondrial A1555G mutation is one of the potential causes of non-syndromic SNHL in the Egyptian population.

A novel WNT10A mutation causes non-syndromic hypodontia in an Egyptian family.

OBJECTIVE: Tooth agenesis is the most common dental anomaly, whose aetiology still remains to be fully elucidated. The aim of this study was to investigate the genetic cause of non-syndromic hypodontia with clinical variability in an Egyptian family. DESIGN: The entire coding regions including exon-intron boundaries of the MSX1, PAX9 and WNT10A genes were investigated by direct sequencing in all affected family members. RESULTS: Novel heterozygous mutation inherited in an autosomal dominant manner was identified in the WNT10A gene. This 21-bp deletion combined with 1-bp insertion, c.-14_7delinsC, eliminates the translation initiation codon leading to either no protein production or translation of alternative open reading frames. None of the control subjects (400 chromosomes) were carriers of this novel WNT10A mutation. No pathogenic mutations were found in the MSX1 and PAX9 genes. CONCLUSIONS: The novel c.-14_7delinsC mutation might be the etiological variant of the WNT10A gene responsible for the permanent tooth agenesis in the Egyptian family. WNT10A is a major candidate gene for non-syndromic hypodontia.

Recurrent hydatidiform mole: detection of two novel mutations in the NLRP7 gene in two Egyptian families.

OBJECTIVES: Hydatidiform mole is an aberrant pregnancy with hyperproliferative vesicular trophoblast and defective fetal development. In 2006, mutations in NLRP7 were found to be responsible for recurrent hydatidiform moles (RHM), but genetic heterogeneity has been demonstrated and mutations of C6orf221 were later reported in several families. Here we report a new Egyptian family in which two sisters had eleven and four molar pregnancies, respectively. The objective was to present the results of the mutation analysis of NLRP7 and C6orf221 genes in Egyptian women with RHM. STUDY DESIGN: Three women from two unrelated Egyptian families; two sisters and a previously described sporadic case, all presenting with RHM, were enrolled. The cases were subjected to detailed history taking, karyotyping and screening for mutations in NLRP7 and C6orf221. RESULTS: Two NLRP7 mutations have been detected, one in each family. In the first family, sequencing identified a homozygous 2 bp deletion in the seventh coding exon of NLRP7, while a homozygous G-to-A substitution in the third coding exon of NLRP7 was detected in the second family. Both of them result in a truncated protein. The two mutations have not been previously described in the literature. No mutations in C6orf221 were found in any of the samples. CONCLUSION: The detection of an NLRP7 mutation in both the familial and the apparently isolated case of RHM provides further evidence for the previously established role of NLRP7 mutations in the pathophysiology of RHM and increases the diversity of mutations described in the Egyptian population. Our results also expand further the spectrum of reproductive wastage associated with NLRP7 mutations to patients with recurrent spontaneous abortion.